Recombinant methods for the production of a bacillus alkaline protease

ABSTRACT

The invention is directed to an isolated nucleic acid construct(s) comprising a nucleic acid sequence encoding an alkaline protease having a molecular weight of about 34 kD and is obtainable from a strain of Bacillus sp. Group I, as well as recombinant vectors and host cells comprising such constructs. Additionally, the invention is directed to a method for obtaining the protease. The invention is also directed to promoter and signal sequences derived from said alkaline protease as well as methods of using the promoter and signal sequences in the expression of heterologous nucleic acid sequences encoding heterologous proteins in bacteria.

This application is a divisional application of co-pending application Ser. No. 08/434,255, filed May 3, 1995, which is continuation-in-part of application Ser. No. 08/325,386, filed Oct. 26, 1994, which is a continuation of PCT/DK93/00183, filed on May 26, 1993, which are incoproated herein by reference.

1. TECHNICAL FIELD

The invention is directed to an isolated nucleic acid construct (s) comprising a nucleic acid sequence encoding a novel alkaline protease derived from a strain of Bacillus sp. Group I, as well as recombinant vectors and host cells comprising such constructs. Additionally, the invention is directed to a method for obtaining the protease. The invention is also directed to promoter and signal sequences derived from said alkaline protease as well as methods of using such promoter and signal sequences in the expression of heterologous nucleic acid sequences encoding heterologous proteins in bacteria.

2. BACKGROUND OF THE INVENTION

Detergent enzymes have been marketed for more than 20 years and are now well established as normal detergent ingredients in both powder and liquid detergents all over the world. With the trend towards lower washing temperature, detergent enzyme consumption has increased during late years. Enzymes used in washing formulations comprise proteases, lipases, amylases, cellulases, as well as other enzymes, or mixtures thereof. Commercially most important are proteases.

Detergent proteases have been developed by isolation of proteases found in nature followed by testing in detergent formulations. Most detergent proteases are obtained from members of the genus Bacillus. Currently, new types of proteases enter the market, offering the possibility of giving a better cost/performance ratio at various specified conditions.

Examples of commercial protease products are ALCALASE™, ESPERASE™ and SAVINASE™, all supplied by Novo Nordisk A/S, Denmark. These and similar enzyme products from other commercial sources are active in detergent solutions, i.e., at pH values in the range of from 8 to 11 and in the presence of sequestering agents, surfactants and bleaching agents such as sodium borate. The ALCALASE™ protease is produced by strains of the species Bacillus licheniformis. The ESPERASE™ and SAVINASE™ proteases are obtained by cultivation of strains of alkalophilic Bacilli.

It would be advantageous to provide novel alkaline proteases with a unique range of substrates, pH and/or temperature optima. It would also be advantageous to isolate novel proteases or produce proteases in high yield so that the proteases could be used in vitro. It would be advantageous to determine the amino acid and/or nucleic acid sequence of these proteases in order to determine, e.g., conserved and nonconserved regions and active sites, and thus, appropriate sites for mutagenesis to improve enzyme performance and to be able to produce the proteases using recombinant DNA technology.

By determining the nucleic acid sequence of the entire protease gene, one may determine the location of promoter and signal sequences. Such sequences may be of use in the expression of heterologous polypeptides.

3. SUMMARY OF THE INVENTION

The invention is related to a nucleic acid construct comprising a nucleic acid sequence encoding a protease having an apparent molecular weight of about 34 kD as determined by SDS-PAGE, a pI of approximately 9.3, a pH optimum in the range of about 9-11 determined at about 25° C. (with casein as substrate), a temperature optimum in the range of about 40°-55° C. determined at about pH 9.5 (with casein as substrate), and being obtainable from a strain of Bacillus sp. of group 1. Bacilius sp. of group 1 are most similar to B. subtilis and B. firmus. In a specific embodiment, the protease of the present invention is obtainable or derived from the PD498 strain, or from another host organism carrying the gene encoding a protease having immunochemical properties substantially identical or partially identical to those of the protease derived from Bacillus sp. PD498.

In one embodiment, the nucleic acid construct comprises the nucleic acid sequence encoding a protease having an amino acid sequence depicted in SEQ ID NO:1, which shows the sequence of the PD498 alkaline protease in its prepro form, SEQ ID NO:2, which shows the alkaline protease in its pro form or SEQ ID NO:3, which shows the PD498 alkaline protease in its mature form. In another embodiment, the nucleic acid construct comprises the nucleic acid sequence depicted in SEQ ID NO:4, which shows the entire nucleic acid sequence of the PD498 alkaline protease gene, which includes the coding region as well as the 5' and 3' flanking regions, SEQ ID NO:5, which shows the nucleic acid sequence encoding the PD498 alkaline protease in its prepro form, SEQ ID NO:6, which shows the nucleic acid sequence encoding the PD498 gene in its pro form, or SEQ ID NO:7, which shows the DNA sequence encoding the PD498 alkaline protease in its mature form.

In order to facilitate production of the novel protease, the invention also provides vectors, and recombinant host cells comprising the claimed nucleic acid construct, which vectors, constructs and recombinant host cells are useful in the recombinant production of the protease. Recombinant production of the protease of the invention is achieved by culturing a host cell transformed or transfected with the nucleic acid construct or vector of the invention, or progeny thereof, under conditions suitable for expression of the protease, and recovering the protease from the culture.

The invention is also directed to a promoter sequence derived from a gene encoding said novel protease or fragment thereof having substantially the same promoter activity as said sequence in which said promoter has the sequence shown in SEQ ID NO:8. The promoter may be used in the construction of a vector comprising said promoter sequence and a site for insertion into said vector of a heterologous DNA sequence. The term "heterologous" is intended to include a DNA sequence not expressed by the host cell in nature. Furthermore, the promoter may be used in a method for producing a heterologous protein comprising (a) transforming a bacterial cell, preferably a gram positive bacterium with said vector; (b) culturing the transformed cell of step (a); and (c) recovering said heterologous polypeptide from the tranformed cell of step (b).

The invention is also directed to a signal sequence derived from a gene encoding said novel protease or fragment thereof having substantially the same signal activity as said sequence in which said signal sequence has the amino acid sequence shown in SEQ ID NO:9. The signal sequence may be encoded by the DNA sequence shown in SEQ ID NO:10. The signal sequence may be used in the construction of a vector comprising (a) said signal sequence; (b) upstream from said signal sequence, a promoter sequence and a ribosome binding site sequence, transcription of said signal sequence being under the control of said promoter sequence; and (c) downstream from said signal sequence, a site for insertion into said vector of a heterologous DNA sequence, in the same reading frame as said signal sequence. Furthermore, the signal sequence may be used in a method for producing a heterologous protein comprising (a) transforming a bacterial cell, preferably a gram positive bacterium with said vector; (b) culturing the transformed cell of step (a); and (c) recovering said heterologous polypeptide from the tranformed cell of step (b).

The protease may be used in the washing process and therefore may be formulated into a detergent additive and/or composition.

4. BRIEF DESCRIPTION OF DRAWINGS

The present invention is further illustrated by reference to the accompanying drawings, in which:

FIG. 1 shows shows the relation between temperature and the proteolytic activity of the PD498 alkaline protease with 2% casein as substrate, determined at pH 9.5; Buffer, Buffer+0.1% STPP (sodium tripolyphosphate).

FIG. 2 shows the relation between pH and the proteolytic activity of the PD498 alkaline protease with 1% casein as substrate, determined at 25° C.

FIGS. 3A through 3I show the complete amino acid and nucleotide sequence of the PD498 protease as well as associated upstream and downstream nucleotide sequences.

FIG. 4 shows the structure of PD498 protease.

FIG. 5 shows the construction of promoter fusions.

FIG. 6 shows a diagram of the plasmid p118-498prom1.

FIG. 7 shows a diagram of pHP13amp.

FIG. 8 shows a diagram of pLip3.

FIG. 9 shows the construction of p498-8.

FIG. 10 shows a diagram of pMHan37.

FIG. 11 shows a diagram of the PD498-lipolase fusion.

5. DETAILED DISCLOSURE OF THE INVENTION

5.1. Isolation of the Protease

5.1.1. The Microorganism

The microorganism which is able to produce said alkaline protease is isolated from a soil sample.

The microorganism is an aerobic, spore forming bacterium belonging to the genus Bacillus, group 1. In a preferred embodiment, morphologically it can be described as a motile rod with a diameter of 0.7-0.9 micron, and a length of 2-3 microns. The spores are round to ellipsoid, slightly swelling the sporangium, subterminal to terminal. Optimum temperature for growth is within 25°-37° C., and optimal pH for growth is within 7-9. No growth occurs at pH 9.7, nor at 50° C. The microorganism forms yellow colonies, round and smooth, on nutrient agar slants, and no diffusion of pigment into the agar is observed. In a specific embodiment, the microorganism is Bacillus sp. PD498 has been deposited according to the Budapest Treaty at NClMB, under No. 40484.

5.1.2. Cultivation of the Microorganism

The microorganism which produces said alkaline protease can be cultivated under aerobic conditions in a nutrient medium containing assimilable carbon and nitrogen together with other essential nutrients, the medium being composed in accordance with the principles of the known art.

Suitable carbon sources are carbohydrates such as sucrose, glucose and starch, or carbohydrate containing materials such as cereal grain, malt, rice and sorghum. The carbohydrate concentration incorporated in the medium may vary widely, e.g., up to about 25% and down to about 1-5%, but usually about 8-10% will be suitable, the percentages being calculated as equivalents of glucose.

The nitrogen source in the nutrient medium may be of inorganic and/or organic nature. Suitable inorganic nitrogen sources are nitrates and ammonium salts. Among the organic nitrogen sources quite a number are used regularly in fermentation processes involving the cultivation of bacteria. Illustrative examples are soybean meal, cotton seed meal, peanut meal, casein, corn, corn steep liquor, yeast extract, urea, and albumin. In addition, the nutrient medium should also contain usual trace substances.

The microorganism is a strain of the Bacillus species. The Bacillus species is slightly alkalophilic. Therefore, the cultivation is preferably conducted at slightly alkaline pH values, which can be obtained by addition of suitable buffers such as sodium bicarbonate, about pH 9.0, after sterilization of the growth medium. For cultivation in tank fermentors, it is necessary to use artificial aeration. The rate of aeration is similar to that used in conventional tank fermentation.

After fermentation, liquid protease concentrates may be produced by removal of coarse material from the broth and, if desired, concentration of the broth by evaporation at low temperature or by ultrafiltration. Finally, preservatives may be added to the concentrate.

Solid protease preparations may be prepared from the purified and/or concentrated broth by precipitation with salts, such as Na₂ SO₄ or water-miscible solvents, such as ethanol or acetone. Removal of the water in the broth by suitable drying methods, such as spray-drying, may also be employed.

5.1.3. Assay for Proteolytic Activity

The proteolytic activity may be determined with casein as substrate. One Casein Protease Unit (CPU) is defined as the amount of protease liberating about 1 μM of primary amino groups (determined by comparison with a serine standard) per minute under standard conditions, i.e., incubation for about 30 minutes at about 25° C. and about pH 9.5.

The proteolytic activity may also be determined by measuring the specific hydrolysis of succinyl-Ala-Ala-Pro-Leu-p-nitroanilide by said protease. The substrate is initially dissolved in for example, DMSO and then diluted about 50 fold in about 0.035M borate buffer, about pH 9.45. All protease samples may be diluted about 5-10 fold by the same borate buffer. Equal volumes of the substrate solution and sample are mixed in a well of an ELISA reader plate and read at about 405 nm at 25° C. All sample activities and concentrations are normalized to the standard protease solution activity and concentration, respectively.

5.2. The Protease

Said protease can be described by the following characteristics.

5.2.1. Physical-Chemical Properties

The protease of the invention has an apparent molecular weight of about 34 kD when determined by SDS-PAGE. A pI of approximately 9.3 is determined by isoelectric focusing on LKB Ampholine® PAG plates. The protease activity is inhibited by PMSF, a serine proteinase inhibitor. EDTA and soybean-protease inhibitor do not influence the protease activity.

The temperature-activity relationship is presented in FIG. 1. The activity is determined with 2% casein as substrate at about pH 9.5 in the presence (white squares) and absence (black squares) of 0.1% sodium tripolyphosphate (STPP, a common ingredient in many commercial detergents). The assay for proteolytic activity described previously is used with the modification that the incubation temperature is varied in the interval of from about 15° to about 70° C. It appears from the figure that the protease possesses proteolytic activity from about 15° C. to about 70° C., and has a temperature optimum in the range of from about 40°-55° C.

The dependence of activity on pH is determined by the same procedure, using buffers adjusted to predetermined pH values in the pH range of from about 6 to 11. The result is shown in FIG. 2. It appears from this figure that the protease possesses proteolytic activity in a very broad pH range of from below about pH 6 to above about pH 11, with an apparent pH optimum in the range of from about pH 9-11.

Furthermore, it is found that the protease of the invention is stable for about 60 minutes at about 25° C. under washing conditions when determined in European type and American type detergents.

5.2.2. Immunochemical Properties

Said protease has immunochemical properties substantially identical or at least partially identical to those of a protease derived from the strain Bacillus sp. PD498, NCIMB No. 40484.

The immunochemical properties can be determined immunologically by cross-reaction identity tests. The identity tests can be performed by the well-known Ouchterlony double immunodiffusion procedure or by tandem crossed immunoelectrophoresis according to N. H. Axelsen; Handbook of Immunoprecipitation-in-Gel Techniques; Blackwell Scientific Publications (1983), chapters 5 and 14. The terms "antigenic identity" and "partial antigenic identity" are described in the same book, Chapters 5, 19 and 20.

Monospecific antiserum is generated according to the above mentioned method by immunizing rabbits with the purified protease of the invention. The immunogen is mixed with Freund's adjuvant and injected subcutaneously into rabbits every second week. Antiserum is obtained after a total immunization period of 8 weeks, and immunoglobulin is prepared therefrom as described by N. H. Axelsen, supra.

Ouchterlony double immunodiffusion tests show no cross reaction between the protease of the invention and the known alkaline serine proteases from Bacillus species, e.g., ALCALASE™, SAVINASE™, ESPERASE™, subtilisin Novo (available from Novo Nordisk A/S, Denmark), and KAZUSASE™ (available from SHOWA DENKO, Japan).

5.3. Nucleic Acid Construct

As used herein the term "nucleic acid construct" is intended to indicate any nucleic acid molecule of genomic DNA, synthetic DNA or RNA origin. The term "construct" is intended to indicate a nucleic acid segment which may be single- or double-stranded, and which map be based on a complete or partial naturally-occurring nucleotide sequence encoding the protease. The construct may optionally contain other nucleic acid segments.

The nucleic acid construct of the invention encoding said protease may suitably be of genomic origin, for instance obtained by preparing a genomic library and screening for DNA sequences coding for all or part of the protease by hybridization using synthetic oligonucleotide probes in accordance with standard techniques (cf. Sambrook et al., Molecular Cloning, A Laboratory Mannual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). For the present purpose, the DNA sequence encoding the protease is obtainable by isolating chromosomal DNA from the strain of Bacillus sp. described in Section 5.1, supra and screening a genomic DNA library.

The nucleic acid construct of the invention encoding the protease may also be prepared synthetically by established standard methods, e.g., the phosphoramidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869, or the method described by Matthes et al., EMBO Journal 3 (1984), 801-805. According to the phosphoramidite method, oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors.

Furthermore, the nucleic acid construct may be of mixed synthetic and genomic origin and may be prepared by ligating fragments of synthetic or genomic DNA (as appropriate), the fragments corresponding to various parts of the entire nucleic acid construct, in accordance with standard techniques.

The nucleic acid construct may also be prepared by polymerase chain reaction using specific primers, for instance as described in U.S. Pat. No. 4,683,202 or Saiki et al., Science 239 (1988), 487-491.

In a currently preferred embodiment, the nucleic acid construct of the invention comprises the DNA sequence shown in SEQ ID NO:4 as well as nucleic acid sequences encoding the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, e.g., DNA sequences depicted in SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7, respectively. The invention further encompasses nucleic acid sequences which hybridize to a nucleic acid molecule (either genomic or synthetic) encoding the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 under the following conditions: presoaking in 5x SSC and prehybridizing for 1 hr. at about 40° C. in a solution of 20% formamide, 50 mM sodium phosphate, pH 6.8, and 50 μg denatured sonicated calf thymus DNA, followed by hybridization at about 40° C. for 18 hrs. in the same solution, followed by a wash in 0.4X SSC at a temperature of about 45° C.

Useful variants within the categories defined above include, for example, ones in which the DNA encodes changes in which conservative amino acid substitutions have been made, which substitutions do not significantly affect the activity of the protein. By conservative substitution is meant that amino acids of the same class may be substituted by any other of that class. For example, the nonpolar aliphatic residues, Ala, Val, Leu, and Ile may be interchanged, as may be the basic residues Lys and Arg, or the acidic residues Asp and Glu. Similarly, Ser and Thr are conservative substitutions for each other, as are Ash and Gln. It will be apparent to the skilled artisan that such substitutions can be made outside the regions critical to the function of the molecule and still result in an active protease. Retention of the desired activity can readily be determined by using the assay procedures described above.

The nucleic acid construct is preferably a DNA construct which term will be used exclusively in the following.

5.4. Recombinant Vector

In a further aspect, the present invention relates to a recombinant vector comprising a DNA construct of the invention. The recombinant vector into which the DNA construct of the invention is inserted may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e., a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector in which the DNA sequence encoding the protease of the invention is operably linked to additional segments required for transcription of the DNA. In general, the expression vector is derived from plasmid or viral DNA, or may contain elements of both. The term, "operably linked" indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in a promoter and proceeds through the DNA sequence coding for the protease of the present invention.

The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Specifically, examples of suitable promoters for use in bacterial host cells include but are not limited to the promoter of the Bacillus stearothermophilus maltogenic amylase gene, the the Bacillus amyloliquefaciens (BAN) amylase gene, the Bacillus subtilis alkaline protease gene, the Bacillus pumilus xylosidase gene, the Bacillus thuringiensis cryIIIA or the Bacillus licheniformis alpha-amylase gene. Other promoters that may be used include but are not limited to phage Lambda P_(R) or P_(L) promoters or the E. coli lac, trp or tac promoters.

The promoter may also be derived from a gene encoding said protease or a fragment thereof having substantially the same promoter activity as said sequence. The sequence of the promoter is shown in SEQ ID NO:8. The invention further encompasses nucleic acid sequences which hybridize to the promoter sequence shown in SEQ ID NO:8 under the following conditions: presoaking in 5X SSC and prehybridizing for 1 hr. at about 40° C. in a solution of 20% formamide, 50 mM sodium phosphate, pH 6.8, and 50 μg denatured sonicated calf thymus DNA, followed by hybridization in the same solution for 18 hrs. at about 40° C., followed by a wash in 0.4X SSC at a temperature of about 45° C., or which have at least about 90% homology and preferably about 95% homology to SEQ ID NO:8, but which have substantially the same promoter activity as said sequence. This promoter may be used to promote the expression of either said protease or a heterologous DNA sequence, e.g. lipolase®, a 1,3-specific lipase, hereinafter referred to as Lipolase®. The enzyme may be encoded by the DNA sequence shown in SEQ ID NO:11 and may have an amino acid sequence shown in SEQ ID NO:12. The enzyme may also be a Lipolase® variant, e.g., D96L, E210K, E210L (see WO 92/05249).

The recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. When the host cell is a bacterial cell, sequences enabling the vector to replicate are various ori sequences.

The vector may also comprise a selectable marker, e.g., a gene the product of which which confers resistance to a drug, e.g., ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, hygromycin or methotrexate.

To direct a protease of the present invention into the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence or pre sequence) may be provided in the recombinant vector. The secretory signal sequence is joined to the-DNA sequence encoding the pro immature protease in the correct reading frame (see, for example, FIG. 4). A pro sequence is an amino acid sequence between the pre sequence and mature protease that is necessary for the secretion of the protease. Cleavage of the pro sequence will result in a mature active protease. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the protease. The secretory signal sequence may be that normally associated with the protease or may be from a gene encoding another secreted protein.

For secretion from bacterial cells, the signal peptide may be a naturally occurring signal peptide, or a functional part thereof, or it may be a synthetic peptide. Suitable signal peptides include but are not limited to sequences derived from Bacillus licheniformis alpha-amylase, Bacillus lentus alkaline protease, and Bacillus amyloliquefaciens amylase.

For secretion from bacterial cells, the signal peptide may also be the signal peptide from the protease disclosed in the instant application. The amino acid sequence of the signal sequence is shown in SEQ ID NO:9. The signal sequence may be encoded by a nucleic acid sequence depicted in SEQ ID NO:10. The invention further encompasses nucleic acid sequences which hybridize to the nucleic acid sequence shown in SEQ ID NO:10 under the following conditions: presoaking in 5X SSC and prehybridizing for 1 hr. at about 40° C. in a solution of 20% formamide, 50 mM sodium phosphate, pH 6.8, and 50 μg denatured sonicated calf thymus DNA, followed by hybridization in the same solution for 18 hrs. at about 40° C., followed by a wash in 0.4X SSC at a temperature of about 45° C., or which have at least about 90% homology and preferably about 95% homology to SEQ ID NO:5, but which have substantially the same signal activity as said sequence. This signal may be used to facilitate the secretion of either said protease or a heterologous DNA sequence, e.g. lipolase®, a 1,3-specific lipase, hereinafter referred to as Lipolase®. The enzyme may be encoded by the DNA sequence shown in SEQ ID NO:11 and may have an amino acid sequence shown in SEQ ID NO:12. The enzyme may also be a Lipolase® variant, e.g., D96L, E210K, E210L (see WO 92/05249).

The procedures used to ligate the DNA sequences coding for the present protease, the promoter and/or secretory signal sequence, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., op. cid.).

5.5. Host cells

The DNA sequence encoding the present alkaline protease introduced into the host cell may be either homologous or heterologous to the host in question. If homologous to the host cell, i.e., produced by the host cell in nature, it may be operably connected to another promoter sequence or, if applicable, another secretory signal sequence and/or terminator sequence than in its natural environment. The term "homologous" is intended to include a nucleic acid sequence encoding a protease native to the host organism in question; the nucleic acid acid sequence may be introduced into the host organism in multicopy form. The term "heterologous" is intended to include a DNA sequence not expressed by the host cell in nature. Thus, the DNA sequence may be from another organism, or it may be a synthetic sequence.

The host cell into which the DNA construct or the recombinant vector of the invention is introduced may be any cell which is capable of producing the present alkaline protease and includes but is not limited to bacteria, yeast, fungi and higher eukaryotic cells.

Examples of bacterial host cells which, on cultivation, are capable of producing and secreting the protease of the invention are gram positive bacteria such as strains of Bacillus, such as strains of B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. megatherium or B. thuringiensis, or gram negative bacteria such as Escherichia coli. The transformation of the bacteria may be effected by protoplast transformation or by using competent cells in a manner known per se (cf. Sambrook et al., supra).

When expressing the protease in bacteria such as E. coli, the protease may be retained in the cytoplasm, typically as insoluble granules (known as inclusion bodies), or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed and the granules are recovered and denatured after which the protease is refolded by diluting the denaturing agent. In the later case, the protease may be recovered from the periplasmic space by disrupting the cells, e.g., by sonication or osmotic shock, to release the contents of the periplasmic space and recovering the protease.

The transformed host cell described above is then cultured in a suitable nutrient medium under conditions permitting the expression of the present protease, after which the resulting protease is recovered from the culture. The medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g., in catalogues of the American Type Culture Collection). The protease produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g., ammonium sulphate, purification by a variety of chromatographic procedures, e.g., ion exchange chromatography, gel filtration chromatography, affinity chromatography, or the like, dependent on the type of protease in question.

The invention is further illustrated in the following examples, which are not intended to be in any way limiting to the scope of the invention as claimed.

6. EXAMPLES

6.1. Isolation of PD498 Protease

Bacillus sp. PD498 is cultivated at 25° C. on a rotary shaking table (300 r.p.m.) in 500 ml baffled Erlenmeyer flasks containing 100 ml of medium of the following composition (per liter):

    ______________________________________                                         Potato starch          100    g                                                Ground barley          50     g                                                Soybean flour          20     g                                                Na.sub.2 HPO.sub.4 x 12H.sub.2 O                                                                      9      g                                                Pluronic 54R (BASF)    0.1    g                                                Sodium caseinate       10     g                                                ______________________________________                                    

The starch in the medium is liquified with alpha-amylase, and the medium is sterilized by heating at 120° C. for 45 minutes.

After sterilization, the pH of the medium is adjusted to 9.0 by addition of 10 ml of a 1M solution of sodium bicarbonate.

After 4 days of incubation, the proteolytic activity of the culture is determined using the method described above. The protease activity of the broth is 5 CPU/l.

After separation of the solid material the protease is purified by a conventional chromatographic method. Yield from 3.5 l of culture broth is 31 ml with 120 CPU/l. Purity is more than 90% as judged by both SDS-PAGE and isoelectric focusing.

The characteristics of the preparation prepared in accordance with this Example have been referred to earlier in this specification, and reference is made hereto.

6.2. Cloning and Expression of PD498 Protease

6.2.1. Materials and Methods

6.2.1.1. Strains

Escherichia coli DH5 F-Φ80dlacZΔ M15Δ(lacZYA-argF) U169 deoR recA1 endA1 hsdR17(r_(K-),m_(K+))supE44-thi-1 gyrA96 relA1 (BRL) is used for all E. coli transformations according to manufacturer's specifications (BRL). A B. subtilis protease deficient strain is made competent by the procedure of Anagnostopolis and Spizizen (J. Bacteriol. 81: 741-746, 1961).

6.2.1.2. DNA Isolation and Gene Library Construction

PD498 chromosomal DNA is isolated as described by Pitcher, et. al. (Letters in Applied Micro. 8: 151-156, 1989). 100 μg of DNA is digested with EcoR1 and run on an agarose gel. DNA of 5-8 kb is isolated using the Qiaex system (Qiagen). This DNA is ligated to EcoR1-cut, CIP (calf intestinal phosphatase) treated pUC19 (BRL) at a ratio of 4:1 respectively. The ligation mix is then transformed into E. coli strain DH5alpha (BRL).

6.2.1.3. PCR Amplification of the 180 bp Fragment

The amplification of a 180 bp fragment of the protease gene is performed using two mixed primers, group A and B, based on partial amino acid sequence of the protease. The letter amino acid sequences are Sequence A: AYQYGPQNT (SEQ ID NO:13) and sequence B: YDFIDYDNNPMD (SEQ ID NO:14). The primers and total PD498 DNA are used in a reaction of 95° C./5 minutes followed by 30 cycles of 95° C./1 minute, 55° C./1 minute, 72° C./1 minute in an Ericomp PCR machine.

    Primer group A: GCNTAYCARTAYGGNCCNCARAAYAC (SEQ ID NO:15)

    Primer group B: TCCATNGGRTTRTTRTCRTARTCDATRAARTCRTA (SEQ ID NO:16)

6.2.1.4. Subcloning of PCR Reaction Product

The PCR product is cloned for sequencing using the TA Cloning vector pCRII (Invitrogen, San Diego, Calif.) following manufacturer's specifications. The insert DNA is DIG (digoxigenin)-labeled and Southern hybridizations and colony blots are performed using the Genius system from Boehringer Mannheim as modified by Engler-Blum (1993, Anal. Biochemistry 210:235-244).

6.2.1.5. DNA Sequencing

DNA sequences are determined using Taq polymerase cycle-sequencing with fluorescent labeled dideoxynucleotides. The sequencing reactions are run on an Applied Biosystems automatic DNA sequencer (Model 373A, version 1.2.0).

6.2.1.6. Construction of Vector Containing PD498 Protease Driven By the amyL Promoter

The alpha-amylase (amyL) promoter (SEQ ID NO:17), an "up" mutant promoter from Bacillus licheniformis, is obtained by PCR amplification from B. licheniformis DNA using the following primers:

    5'-AAGGCATGCGTCCTTC-3' (SEQ ID NO:18)

    5'-CTTTCAATGTGTAACATA-3' (SEQ ID NO:19)

The resulting 630 bp fragment is cloned into the pCRII vector (Invitrogen), enabling the cloned fragment to be easily isolated as an EcoRI fragment.

The protease gene is subcloned as a 1.9 kb EcoRI-EcoRV fragment into EcoRI, SmaI-cut pUC19, then removed as a 1.9 kb EcoRI-HindIII fragment and cloned into EcoRI-HindIII-cut pHP13amp, an E. coli-B. Subtilis shuttle vector (FIG. 8). The resulting plasmid, p498-4, is digested with EcoRI, treated with calf intestinal phosphatase (CIP), and ligated to the amyL promoter fragment described above, generating plasmid p498-5 (FIG. 5). B. subtilis colonies containing the promoter in the correct orientation relative to the PD498 coding region are selected by their ability to form halos on agar plates containing the appropriate antibiotic and 1% milk.

6.2.1.7. Construction of vector Containing PD498 Protease Driven By cryIIIA Promoter

The cryIIIA promoter (SEQ ID NO:20) from Bacillus thuringiensis var. tenebrionis is amplified by PCR using the following primers:

    5'-GAGACCCGGGAGCTTTCAGTGAAGTACGTG-3' (SEQ ID NO:21)

    5'-CATAAATCCATTAGACGGTGC-3' (SEQ ID NO:22)

The resulting 1500 bp fragment is again cloned into the pCRII vector (Invitrogen), removed as an EcoRI fragment, and ligated to EcoRI-digested p498-4 plasmid to generate plasmid p498-6, where PD498 protease expression is driven by the cryIIIA promoter (FIG. 5). B. subtilis colonies containing the promoter in the correct orientation relative to the PD498 coding region are selected by their ability to form halos on agar plates containing the appropriate antibiotic and 1% milk.

6.2.1.8. Construction of Vector Containing PD498 Protease Driven By The BAN Promoter

The Bacillus amyloliquefaciens amylase (BAN) promoter is PCR-amplified from a plasmid (pSX222) (SEQ ID NO:23) using an upstream primer containing an additional SphI linker,

    5'-GCATGCAATCGATTGTTTGAGAAAAGAAG-3', (SEQ ID NO:24)

where the first set of underlined nucleotide sequences is the said SphI site and the second set of underlined nucleotide sequences is a naturally occurring ClaI site; and a downstream primer,

    5'-CATTTTCTTATACAAATTATATTTTACATATCAG-3' (SEQ ID NO:25).

The resulting 168 bp fragment is cloned into the pCRII vector (Invitrogen), removed as an EcoRI fragment, and ligated to EcoRI-digested p498-4 plasmid to generate plasmid p498-7, where PD498 protease expression is driven by the BAN promoter (FIG. 5). B. subtilis colonies containing the promoter in the correct orientation relative to the PD498 coding region are selected by their ability to form halos on agar plates containing the appropriate antibiotic and 1% milk.

B. subtilis transformants containing the BAN promoter in the reverse orientation relative to the PD498 protease are selected by their inability to form halos on agar plates containing the appropriate antibiotic and 1% milk.

6.2.2. Sequence Analysis of the PD498 Gene

Using two groups of oligonucleotides based on partial amino acid sequence of the PD498 protease, the correct sized fragment of 180 bp is amplified by PCR and cloned into the E. coli vector pCRII. Sequencing of the 180 bp insert reveals that the correct fragment is cloned. Using the 180 bp insert as a probe, Southern hybridizations identify a 6.5 kb EcoR1 fragment from PD498 chromosomal DNA that hybridize to the probe. Chromosomal DNA from PD498 is digested with EcoR1 and fragments of 5-8 kb are size-selected and ligated into pUC19 that is cut with EcoR1 and treated with CIP. Thousands of white colonies result after transformation into E. coli strain DH5α selecting on LB plates containing Amp and X-gal. 2 of 600 colonies screened are positive by colony hybridization using the labeled 180 bp fragment as a probe. Restriction analyses indicate that both colonies contain identical plasmids with a 6.5 kb EcoR1 insert.

DNA sequencing indicates that the 5' EcoR1 site is just upstream of the genes ribosome binding site (FIG. 3; SEQ ID NO:26) followed by the ATG initiation codon of the protein. The first 27 amino acids resemble a typical Bacillus signal peptide with a short sequence containing three positively charged residues followed by a hydrophobic stretch ending with Ser-Leu-Ala. After this cleavage site, there is a propeptide of 90 amino acids followed by the mature protein of 280 amino acids with a predicted molecular weight of 29270, which is in close agreement with the observed molecular weight of 34,000 (FIG. 4) on SDS-PAGE.

6.3. Isolation of a DNA Sequence Upstream of the PD498 Open Reading Frame With Promoter Activity

DNA sequencing of the PD498 coding region indicates the presence of Asp718 (site also recognized by KpnI), PstI, BglII, and other unique restriction sites. Digestion of PD498 genomic DNA with Asp718 and a variety of other enzymes, Southern transfer of the fragments to nylon, and detection with the 180 bp DIG-labeled DNA probe (described in sections 6.2.1.3 and 6.2.1.4), indicates the presence of a 1.4 kb HindIII-Asp718 fragment. From the location of the known Asp718 restriction site in the PD498 coding region, the 1.4 kb HindIII-Asp718 fragment should contain approx 750-850 nts upstream of the ATG translational start of the PD498 protease. A size-selected library of 1-2 kb HindIII-Asp718 fragments is cloned into pUC118 (Vieira and Messing, Methods Enzymol. 153:3-11, 1987) and transformed into E. coli XL1-Blue MRF' cells (Stratagene, San Diego, Calif.). Colonies are screened with the 180 bp DIG-labeled PCR fragment by the colony hybridization technique of Sambrook et al., 1989, supra. Six colonies are detected with the probe, and plasmid DNA is isolated from 4 colonies by the alkaline lysis technique (Sambrook et al., 1989 supra). All four plasmid preparations contain a 1.4 kb HindIII-Asp718 fragment. The sequence of this fragment is determined by Taq polymerase cycle-sequencing with fluorescent-labeled dideoxynucleotides on an ABI 373A sequencer (SEQ ID NO:4). The plasmid containing the fragment is termed p118-498prom1 (see FIG. 6).

6.4. Reconstruction of the PD498 Promoter with the PD498 Coding Sequence

The upstream HindIII-Asp718 fragment is reconstituted with its native PD498 coding sequence in three steps (see FIG. 9). First, the HindIII-Asp718 fragment from p118-498prom1 is cloned into pHP13amp. Then, plasmid p498-5 is digested with MscI and BamHI to yield a 1.0 kb MscI-BamHI fragment containing the coding region of the PD498 protease. Third, the MscI-BamHI fragment is ligated into the plasmid of step one cut with MscI and BamHI. The ligation mixture is then transformed into E. coli DH5α cells. The desired construct, p498-8 (see FIG. 9), is isolated from transformants by alkaline lysis "miniprepping". The plasmid is then transformed into a B. subtilis protease deficient strain and plated on TBAB plates containing 1% non-fat dry milk and 10 μg chloramphenicol per ml. Expression of the protease from its native promoter is indicated by the formation of clear zones (halos) around each transformed B. subtilis colony.

6.5. PD498

Shake flask experiments are performed in a sucrose/soybean flour medium with a B. subtilis protease deficient strain transformed with p498-5, 6, 7, 8, and "7R". These plasmid constructs consist of the pHP13amp vector containing the amyL, cryIIIA, BAN, 498, and reverse BAN (BAN promoter in reverse orientation) respectively, driving PD498 protease expression. B. licheniformis alpha-amylase under the control of cryIIIA, BAN, and PD498 promoters is produced after four days.

6.6. Lipolase Expression in Bacillus subtilis

The following contructs are tested in a B. subtilis protease deficient background: (promoter/signal sequence/Lipolase®)

    ______________________________________                                                    Lipolase ® Activity                                             ______________________________________                                         pHP13amp     0                                                                 pLip3        5                                                                 ______________________________________                                    

A diagram of pHP13amp is shown in FIG. 7 and a diagram of pLip3 is shown in FIG. 8. The following procedure is used to consruct pLip3. The MscI-BamHI fragment of pMHan37 (see FIG. 10), bearing the proLipolase®coding sequence, is ligated with the HindIII-MscI fragment of PD498 from p118-498prom1 (comprising the promoter and signal peptide coding sequence) and HindIII/BamHI-cut pHP13 amp to produce pLip3. A protease deficient B. subtilis strain is transformed with this consturct. When patched on TBAB plates containg 5 ug chloramphenicol/ml and 1% tributyrin, transformants make larger zones of clearing than the B. subtilis protease deficient strain contiang pHP13amp alone. A diagram of the fusion is shown in FIG. 11.

These results are obtained from shake flasks using a sucrose-soy media. The data show that the pD498 promoter/signal sequence can be used to express a heterologous protein in B. subtilis.

7 . DEPOSIT OF MICROORGANISMS

The following biological materials have been deposited in the Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 University Street, Peoria, Ill., 61604, U.S.A.

    ______________________________________                                         Strain            Accession No.                                                                              Deposit Date                                     ______________________________________                                         E. coli containing p498-5                                                                        NRRL B-21434                                                                               April 24, 1995                                   E. coli containing p118-498 prom1                                                                NRRL B-21433                                                                               April 24, 1995                                   ______________________________________                                    

The strains have been deposited under conditions that assure that access to the culture will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C. §122 and under conditions of the Budapest Treaty. The deposit represents a biologically pure culture of each deposited strain. The deposit is available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 27                                                  (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2702 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 843..2033                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        AAGCTTAATCATCCCGATGTATCGCTTCAGCGCTTACCGTAGACGATTTTCTTTATAGTC60                 TCGATGGATAAAAAGTACCTATCTGAAATGGAAGCAATCGACTCTCCTCCAGTAAAGGCT120                TCAAGGATCGACGTGTTTCTCCGTTTAAGCGCTTCCCTTCCTCCTGACTTTGAGCCCCAG180                GCATTATACTCTTCTTTTCGCTTTGGGATATAAATGGTTTGGCCTTGAACATAGTTTTGT240                AATTCTCTCATTAAGTAATCAGGAATTTGATTTACTGCTTTCAATCGTGTCAGCTCCTTA300                TCATTATTGGATCAATAAGGGACAAAGCCGACATATGAATGGCGATTCATCTAAAACTAC360                CACCCCATGCAAAGGATCGCCGAATCATACAGGCTTTGCATGAGATGCTGCAGATTTCGG420                AAAACGGATTTCCATATGATCACCTCCTAGTATCAGTATACTGATACTAGCAGAAAGATT480                TCCATAAGAATTTCTTATAGTTACCATAATATTATTATATAAACCTACTATATTTGATTT540                TCAATTTGAGAAAATAAGTGACCATTCACCTATCCTTAAAGTTGCTCAACCCCATACAAT600                CATGAAACTTTTCATGCCAAACGTTCATTATGCGAAATCTATCAAAAACTGAGAGTGAAT660                TCATTTTTTGATAGAAAATTAAAACTATTCAATATTTTGTCACAACCTGCTAGAATCCTA720                GGTAATAAGGGTCCCCTACATATCTATCATTCATCACAATGACCTTTGTTCATCTTGAAT780                TCTGAAGGGAGGATCGACCTGCTAATTTGTCGTAAAAAAATAGAAAATGGAGGAATGCTT840                TTATGAAGTTCAAAAAAATAGCCGCTCTATCCTTAGCAACTTCCCTT887                             MetLysPheLysLysIleAlaAlaLeuSerLeuAlaThrSerLeu                                  151015                                                                         GCTTTATTCCCTGCCTTCGGAGGTAGTTCACTGGCCAAGGAAGCACCG935                            AlaLeuPheProAlaPheGlyGlySerSerLeuAlaLysGluAlaPro                               202530                                                                         AAACCGTTCCAACCTATCAACAAAACTTTGGATAAAGGCGCTTTCGAA983                            LysProPheGlnProIleAsnLysThrLeuAspLysGlyAlaPheGlu                               354045                                                                         TCCGGTGAAGTCATCGTCAAATTCAAAGATGGTGTATCCAAAAAGGCA1031                           SerGlyGluValIleValLysPheLysAspGlyValSerLysLysAla                               505560                                                                         CAAGGTTCTGCTCTGAACAAAGCGGAGGCAAATGAACAGAAAGCATCA1079                           GlnGlySerAlaLeuAsnLysAlaGluAlaAsnGluGlnLysAlaSer                               657075                                                                         GCAAAAGATCCATTTCAGGTATTGGAAGTAGCGGACGTCGATCAAGCT1127                           AlaLysAspProPheGlnValLeuGluValAlaAspValAspGlnAla                               80859095                                                                       GTTAAAGCACTGGAAAACAATCCGAATGTAGAATATGCTGAACCAAAC1175                           ValLysAlaLeuGluAsnAsnProAsnValGluTyrAlaGluProAsn                               100105110                                                                      TATACCTTCCAAGCGACTTGGTCACCGAATGACCCTTACTATTCTGCT1223                           TyrThrPheGlnAlaThrTrpSerProAsnAspProTyrTyrSerAla                               115120125                                                                      TACCAGTATGGACCACAAAACACCTCAACCCCTGCTGCCTGGGATGTA1271                           TyrGlnTyrGlyProGlnAsnThrSerThrProAlaAlaTrpAspVal                               130135140                                                                      ACCCGTGGAAGCAGCACTCAAACGGTGGCGGTCCTTGATTCCGGAGTG1319                           ThrArgGlySerSerThrGlnThrValAlaValLeuAspSerGlyVal                               145150155                                                                      GATTATAACCACCCTGATCTTGCAAGAAAAGTAATAAAAGGGTACGAC1367                           AspTyrAsnHisProAspLeuAlaArgLysValIleLysGlyTyrAsp                               160165170175                                                                   TTTATCGACAGGGACAATAACCCAATGGATCTTAACGGACATGGTACC1415                           PheIleAspArgAspAsnAsnProMetAspLeuAsnGlyHisGlyThr                               180185190                                                                      CATGTTGCCGGTACTGTTGCTGCTGATACGAACAATGGAATTGGCGTA1463                           HisValAlaGlyThrValAlaAlaAspThrAsnAsnGlyIleGlyVal                               195200205                                                                      GCCGGTATGGCACCAGATACGAAGATCCTTGCCGTACGGGTCCTTGAT1511                           AlaGlyMetAlaProAspThrLysIleLeuAlaValArgValLeuAsp                               210215220                                                                      GCCAATGGAAGTGGCTCACTTGACAGCATTGCCTCAGGTATCCGCTAT1559                           AlaAsnGlySerGlySerLeuAspSerIleAlaSerGlyIleArgTyr                               225230235                                                                      GCTGCTGATCAAGGGGCAAAGGTACTCAACCTCTCCCTTGGTTGCGAA1607                           AlaAlaAspGlnGlyAlaLysValLeuAsnLeuSerLeuGlyCysGlu                               240245250255                                                                   TGCAACTCCACAACTCTTAAGAGTGCCGTCGACTATGCATGGAACAAA1655                           CysAsnSerThrThrLeuLysSerAlaValAspTyrAlaTrpAsnLys                               260265270                                                                      GGAGCTGTAGTCGTTGCTGCTGCAGGGAATGACAATGTATCCCGTACA1703                           GlyAlaValValValAlaAlaAlaGlyAsnAspAsnValSerArgThr                               275280285                                                                      TTCCAACCAGCTTCTTACCCTAATGCCATTGCAGTAGGTGCCATTGAC1751                           PheGlnProAlaSerTyrProAsnAlaIleAlaValGlyAlaIleAsp                               290295300                                                                      TCCAATGATCGAAAAGCATCATTCTCCAATTACGGAACGTGGGTGGAT1799                           SerAsnAspArgLysAlaSerPheSerAsnTyrGlyThrTrpValAsp                               305310315                                                                      GTCACTGCTCCAGGTGTGAACATAGCATCAACCGTTCCGAATAATGGC1847                           ValThrAlaProGlyValAsnIleAlaSerThrValProAsnAsnGly                               320325330335                                                                   TACTCCTACATGTCTGGTACGTCCATGGCATCCCCTCACGTGGCCGGT1895                           TyrSerTyrMetSerGlyThrSerMetAlaSerProHisValAlaGly                               340345350                                                                      TTGGCTGCTTTGTTGGCAAGTCAAGGTAAGAATAACGTACAAATCCGC1943                           LeuAlaAlaLeuLeuAlaSerGlnGlyLysAsnAsnValGlnIleArg                               355360365                                                                      CAGGCCATTGAGCAAACCGCCGATAAGATCTCTGGCACTGGAACAAAC1991                           GlnAlaIleGluGlnThrAlaAspLysIleSerGlyThrGlyThrAsn                               370375380                                                                      TTCAAGTATGGTAAAATCAACTCAAACAAAGCTGTAAGATAC2033                                 PheLysTyrGlyLysIleAsnSerAsnLysAlaValArgTyr                                     385390395                                                                      TAATAGATAAAACAAGAGCACACCGTGAATGGTGGGCTCTTTCATTATGTTCACTACTGT2093               TTTACGATCTGGCCGTTTTGGTTCAGGTAAACACTCTGGATGATGGTTCTATTAAACGGT2153               TTCCCTTTATAATCAGACTTAATATCCGTTGTCAGGTTGTAGGTTCCTTCTCCTCCATTG2213               AACACTGTACCACTCCCCTTGACAGACTGGGACAAAGGTTTCCCCTTAGGGTAGAACTCA2273               AACATTGTGTGCTCGGTGAACCCACTGACGATACTTGATTGAACGCTGACTCCCTTCTCA2333               GTGGTCGTTACCACCAAGTCATCATTCAATGGACTTGTGAAACCAACATTCAGTAAATAT2393               GCCCCAGGTTCTTTTGACAAAGATGACACCTTCCACTCGCCTTCAATAGGGTTTTCAACC2453               GTTCCCACATGATGAAACGCACCTTTGAAATAACTTTCCTGATCCTTTCCAGATGGTTTC2513               AGTGCCGTTACCTTCCCATCTGGGCTTGTAAGGTAGACATCTTCCTTCGAGTTCGATGCC2573               AACCAGTCAATCGAAATCCGTTCTGCCCCAACCTCTACCCAGAAAGTTTCATCCGCATGC2633               TCTTTATACTCACCTCCGCGGATGAAGGATGAAGTATTGGTCTTGAGAGCCGATTCCTTC2693               CTTGATATC2702                                                                  (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 397 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        MetLysPheLysLysIleAlaAlaLeuSerLeuAlaThrSerLeuAla                               151015                                                                         LeuPheProAlaPheGlyGlySerSerLeuAlaLysGluAlaProLys                               202530                                                                         ProPheGlnProIleAsnLysThrLeuAspLysGlyAlaPheGluSer                               354045                                                                         GlyGluValIleValLysPheLysAspGlyValSerLysLysAlaGln                               505560                                                                         GlySerAlaLeuAsnLysAlaGluAlaAsnGluGlnLysAlaSerAla                               65707580                                                                       LysAspProPheGlnValLeuGluValAlaAspValAspGlnAlaVal                               859095                                                                         LysAlaLeuGluAsnAsnProAsnValGluTyrAlaGluProAsnTyr                               100105110                                                                      ThrPheGlnAlaThrTrpSerProAsnAspProTyrTyrSerAlaTyr                               115120125                                                                      GlnTyrGlyProGlnAsnThrSerThrProAlaAlaTrpAspValThr                               130135140                                                                      ArgGlySerSerThrGlnThrValAlaValLeuAspSerGlyValAsp                               145150155160                                                                   TyrAsnHisProAspLeuAlaArgLysValIleLysGlyTyrAspPhe                               165170175                                                                      IleAspArgAspAsnAsnProMetAspLeuAsnGlyHisGlyThrHis                               180185190                                                                      ValAlaGlyThrValAlaAlaAspThrAsnAsnGlyIleGlyValAla                               195200205                                                                      GlyMetAlaProAspThrLysIleLeuAlaValArgValLeuAspAla                               210215220                                                                      AsnGlySerGlySerLeuAspSerIleAlaSerGlyIleArgTyrAla                               225230235240                                                                   AlaAspGlnGlyAlaLysValLeuAsnLeuSerLeuGlyCysGluCys                               245250255                                                                      AsnSerThrThrLeuLysSerAlaValAspTyrAlaTrpAsnLysGly                               260265270                                                                      AlaValValValAlaAlaAlaGlyAsnAspAsnValSerArgThrPhe                               275280285                                                                      GlnProAlaSerTyrProAsnAlaIleAlaValGlyAlaIleAspSer                               290295300                                                                      AsnAspArgLysAlaSerPheSerAsnTyrGlyThrTrpValAspVal                               305310315320                                                                   ThrAlaProGlyValAsnIleAlaSerThrValProAsnAsnGlyTyr                               325330335                                                                      SerTyrMetSerGlyThrSerMetAlaSerProHisValAlaGlyLeu                               340345350                                                                      AlaAlaLeuLeuAlaSerGlnGlyLysAsnAsnValGlnIleArgGln                               355360365                                                                      AlaIleGluGlnThrAlaAspLysIleSerGlyThrGlyThrAsnPhe                               370375380                                                                      LysTyrGlyLysIleAsnSerAsnLysAlaValArgTyr                                        385390395                                                                      (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1191 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..1191                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        ATGAAGTTCAAAAAAATAGCCGCTCTATCCTTAGCAACTTCCCTTGCT48                             MetLysPheLysLysIleAlaAlaLeuSerLeuAlaThrSerLeuAla                               400405410                                                                      TTATTCCCTGCCTTCGGAGGTAGTTCACTGGCCAAGGAAGCACCGAAA96                             LeuPheProAlaPheGlyGlySerSerLeuAlaLysGluAlaProLys                               415420425                                                                      CCGTTCCAACCTATCAACAAAACTTTGGATAAAGGCGCTTTCGAATCC144                            ProPheGlnProIleAsnLysThrLeuAspLysGlyAlaPheGluSer                               430435440445                                                                   GGTGAAGTCATCGTCAAATTCAAAGATGGTGTATCCAAAAAGGCACAA192                            GlyGluValIleValLysPheLysAspGlyValSerLysLysAlaGln                               450455460                                                                      GGTTCTGCTCTGAACAAAGCGGAGGCAAATGAACAGAAAGCATCAGCA240                            GlySerAlaLeuAsnLysAlaGluAlaAsnGluGlnLysAlaSerAla                               465470475                                                                      AAAGATCCATTTCAGGTATTGGAAGTAGCGGACGTCGATCAAGCTGTT288                            LysAspProPheGlnValLeuGluValAlaAspValAspGlnAlaVal                               480485490                                                                      AAAGCACTGGAAAACAATCCGAATGTAGAATATGCTGAACCAAACTAT336                            LysAlaLeuGluAsnAsnProAsnValGluTyrAlaGluProAsnTyr                               495500505                                                                      ACCTTCCAAGCGACTTGGTCACCGAATGACCCTTACTATTCTGCTTAC384                            ThrPheGlnAlaThrTrpSerProAsnAspProTyrTyrSerAlaTyr                               510515520525                                                                   CAGTATGGACCACAAAACACCTCAACCCCTGCTGCCTGGGATGTAACC432                            GlnTyrGlyProGlnAsnThrSerThrProAlaAlaTrpAspValThr                               530535540                                                                      CGTGGAAGCAGCACTCAAACGGTGGCGGTCCTTGATTCCGGAGTGGAT480                            ArgGlySerSerThrGlnThrValAlaValLeuAspSerGlyValAsp                               545550555                                                                      TATAACCACCCTGATCTTGCAAGAAAAGTAATAAAAGGGTACGACTTT528                            TyrAsnHisProAspLeuAlaArgLysValIleLysGlyTyrAspPhe                               560565570                                                                      ATCGACAGGGACAATAACCCAATGGATCTTAACGGACATGGTACCCAT576                            IleAspArgAspAsnAsnProMetAspLeuAsnGlyHisGlyThrHis                               575580585                                                                      GTTGCCGGTACTGTTGCTGCTGATACGAACAATGGAATTGGCGTAGCC624                            ValAlaGlyThrValAlaAlaAspThrAsnAsnGlyIleGlyValAla                               590595600605                                                                   GGTATGGCACCAGATACGAAGATCCTTGCCGTACGGGTCCTTGATGCC672                            GlyMetAlaProAspThrLysIleLeuAlaValArgValLeuAspAla                               610615620                                                                      AATGGAAGTGGCTCACTTGACAGCATTGCCTCAGGTATCCGCTATGCT720                            AsnGlySerGlySerLeuAspSerIleAlaSerGlyIleArgTyrAla                               625630635                                                                      GCTGATCAAGGGGCAAAGGTACTCAACCTCTCCCTTGGTTGCGAATGC768                            AlaAspGlnGlyAlaLysValLeuAsnLeuSerLeuGlyCysGluCys                               640645650                                                                      AACTCCACAACTCTTAAGAGTGCCGTCGACTATGCATGGAACAAAGGA816                            AsnSerThrThrLeuLysSerAlaValAspTyrAlaTrpAsnLysGly                               655660665                                                                      GCTGTAGTCGTTGCTGCTGCAGGGAATGACAATGTATCCCGTACATTC864                            AlaValValValAlaAlaAlaGlyAsnAspAsnValSerArgThrPhe                               670675680685                                                                   CAACCAGCTTCTTACCCTAATGCCATTGCAGTAGGTGCCATTGACTCC912                            GlnProAlaSerTyrProAsnAlaIleAlaValGlyAlaIleAspSer                               690695700                                                                      AATGATCGAAAAGCATCATTCTCCAATTACGGAACGTGGGTGGATGTC960                            AsnAspArgLysAlaSerPheSerAsnTyrGlyThrTrpValAspVal                               705710715                                                                      ACTGCTCCAGGTGTGAACATAGCATCAACCGTTCCGAATAATGGCTAC1008                           ThrAlaProGlyValAsnIleAlaSerThrValProAsnAsnGlyTyr                               720725730                                                                      TCCTACATGTCTGGTACGTCCATGGCATCCCCTCACGTGGCCGGTTTG1056                           SerTyrMetSerGlyThrSerMetAlaSerProHisValAlaGlyLeu                               735740745                                                                      GCTGCTTTGTTGGCAAGTCAAGGTAAGAATAACGTACAAATCCGCCAG1104                           AlaAlaLeuLeuAlaSerGlnGlyLysAsnAsnValGlnIleArgGln                               750755760765                                                                   GCCATTGAGCAAACCGCCGATAAGATCTCTGGCACTGGAACAAACTTC1152                           AlaIleGluGlnThrAlaAspLysIleSerGlyThrGlyThrAsnPhe                               770775780                                                                      AAGTATGGTAAAATCAACTCAAACAAAGCTGTAAGATAC1191                                    LysTyrGlyLysIleAsnSerAsnLysAlaValArgTyr                                        785790                                                                         (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 397 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        MetLysPheLysLysIleAlaAlaLeuSerLeuAlaThrSerLeuAla                               151015                                                                         LeuPheProAlaPheGlyGlySerSerLeuAlaLysGluAlaProLys                               202530                                                                         ProPheGlnProIleAsnLysThrLeuAspLysGlyAlaPheGluSer                               354045                                                                         GlyGluValIleValLysPheLysAspGlyValSerLysLysAlaGln                               505560                                                                         GlySerAlaLeuAsnLysAlaGluAlaAsnGluGlnLysAlaSerAla                               65707580                                                                       LysAspProPheGlnValLeuGluValAlaAspValAspGlnAlaVal                               859095                                                                         LysAlaLeuGluAsnAsnProAsnValGluTyrAlaGluProAsnTyr                               100105110                                                                      ThrPheGlnAlaThrTrpSerProAsnAspProTyrTyrSerAlaTyr                               115120125                                                                      GlnTyrGlyProGlnAsnThrSerThrProAlaAlaTrpAspValThr                               130135140                                                                      ArgGlySerSerThrGlnThrValAlaValLeuAspSerGlyValAsp                               145150155160                                                                   TyrAsnHisProAspLeuAlaArgLysValIleLysGlyTyrAspPhe                               165170175                                                                      IleAspArgAspAsnAsnProMetAspLeuAsnGlyHisGlyThrHis                               180185190                                                                      ValAlaGlyThrValAlaAlaAspThrAsnAsnGlyIleGlyValAla                               195200205                                                                      GlyMetAlaProAspThrLysIleLeuAlaValArgValLeuAspAla                               210215220                                                                      AsnGlySerGlySerLeuAspSerIleAlaSerGlyIleArgTyrAla                               225230235240                                                                   AlaAspGlnGlyAlaLysValLeuAsnLeuSerLeuGlyCysGluCys                               245250255                                                                      AsnSerThrThrLeuLysSerAlaValAspTyrAlaTrpAsnLysGly                               260265270                                                                      AlaValValValAlaAlaAlaGlyAsnAspAsnValSerArgThrPhe                               275280285                                                                      GlnProAlaSerTyrProAsnAlaIleAlaValGlyAlaIleAspSer                               290295300                                                                      AsnAspArgLysAlaSerPheSerAsnTyrGlyThrTrpValAspVal                               305310315320                                                                   ThrAlaProGlyValAsnIleAlaSerThrValProAsnAsnGlyTyr                               325330335                                                                      SerTyrMetSerGlyThrSerMetAlaSerProHisValAlaGlyLeu                               340345350                                                                      AlaAlaLeuLeuAlaSerGlnGlyLysAsnAsnValGlnIleArgGln                               355360365                                                                      AlaIleGluGlnThrAlaAspLysIleSerGlyThrGlyThrAsnPhe                               370375380                                                                      LysTyrGlyLysIleAsnSerAsnLysAlaValArgTyr                                        385390395                                                                      (2) INFORMATION FOR SEQ ID NO:5:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1110 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..1110                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                        AAGGAAGCACCGAAACCGTTCCAACCTATCAACAAAACTTTGGATAAA48                             LysGluAlaProLysProPheGlnProIleAsnLysThrLeuAspLys                               400405410                                                                      GGCGCTTTCGAATCCGGTGAAGTCATCGTCAAATTCAAAGATGGTGTA96                             GlyAlaPheGluSerGlyGluValIleValLysPheLysAspGlyVal                               415420425                                                                      TCCAAAAAGGCACAAGGTTCTGCTCTGAACAAAGCGGAGGCAAATGAA144                            SerLysLysAlaGlnGlySerAlaLeuAsnLysAlaGluAlaAsnGlu                               430435440445                                                                   CAGAAAGCATCAGCAAAAGATCCATTTCAGGTATTGGAAGTAGCGGAC192                            GlnLysAlaSerAlaLysAspProPheGlnValLeuGluValAlaAsp                               450455460                                                                      GTCGATCAAGCTGTTAAAGCACTGGAAAACAATCCGAATGTAGAATAT240                            ValAspGlnAlaValLysAlaLeuGluAsnAsnProAsnValGluTyr                               465470475                                                                      GCTGAACCAAACTATACCTTCCAAGCGACTTGGTCACCGAATGACCCT288                            AlaGluProAsnTyrThrPheGlnAlaThrTrpSerProAsnAspPro                               480485490                                                                      TACTATTCTGCTTACCAGTATGGACCACAAAACACCTCAACCCCTGCT336                            TyrTyrSerAlaTyrGlnTyrGlyProGlnAsnThrSerThrProAla                               495500505                                                                      GCCTGGGATGTAACCCGTGGAAGCAGCACTCAAACGGTGGCGGTCCTT384                            AlaTrpAspValThrArgGlySerSerThrGlnThrValAlaValLeu                               510515520525                                                                   GATTCCGGAGTGGATTATAACCACCCTGATCTTGCAAGAAAAGTAATA432                            AspSerGlyValAspTyrAsnHisProAspLeuAlaArgLysValIle                               530535540                                                                      AAAGGGTACGACTTTATCGACAGGGACAATAACCCAATGGATCTTAAC480                            LysGlyTyrAspPheIleAspArgAspAsnAsnProMetAspLeuAsn                               545550555                                                                      GGACATGGTACCCATGTTGCCGGTACTGTTGCTGCTGATACGAACAAT528                            GlyHisGlyThrHisValAlaGlyThrValAlaAlaAspThrAsnAsn                               560565570                                                                      GGAATTGGCGTAGCCGGTATGGCACCAGATACGAAGATCCTTGCCGTA576                            GlyIleGlyValAlaGlyMetAlaProAspThrLysIleLeuAlaVal                               575580585                                                                      CGGGTCCTTGATGCCAATGGAAGTGGCTCACTTGACAGCATTGCCTCA624                            ArgValLeuAspAlaAsnGlySerGlySerLeuAspSerIleAlaSer                               590595600605                                                                   GGTATCCGCTATGCTGCTGATCAAGGGGCAAAGGTACTCAACCTCTCC672                            GlyIleArgTyrAlaAlaAspGlnGlyAlaLysValLeuAsnLeuSer                               610615620                                                                      CTTGGTTGCGAATGCAACTCCACAACTCTTAAGAGTGCCGTCGACTAT720                            LeuGlyCysGluCysAsnSerThrThrLeuLysSerAlaValAspTyr                               625630635                                                                      GCATGGAACAAAGGAGCTGTAGTCGTTGCTGCTGCAGGGAATGACAAT768                            AlaTrpAsnLysGlyAlaValValValAlaAlaAlaGlyAsnAspAsn                               640645650                                                                      GTATCCCGTACATTCCAACCAGCTTCTTACCCTAATGCCATTGCAGTA816                            ValSerArgThrPheGlnProAlaSerTyrProAsnAlaIleAlaVal                               655660665                                                                      GGTGCCATTGACTCCAATGATCGAAAAGCATCATTCTCCAATTACGGA864                            GlyAlaIleAspSerAsnAspArgLysAlaSerPheSerAsnTyrGly                               670675680685                                                                   ACGTGGGTGGATGTCACTGCTCCAGGTGTGAACATAGCATCAACCGTT912                            ThrTrpValAspValThrAlaProGlyValAsnIleAlaSerThrVal                               690695700                                                                      CCGAATAATGGCTACTCCTACATGTCTGGTACGTCCATGGCATCCCCT960                            ProAsnAsnGlyTyrSerTyrMetSerGlyThrSerMetAlaSerPro                               705710715                                                                      CACGTGGCCGGTTTGGCTGCTTTGTTGGCAAGTCAAGGTAAGAATAAC1008                           HisValAlaGlyLeuAlaAlaLeuLeuAlaSerGlnGlyLysAsnAsn                               720725730                                                                      GTACAAATCCGCCAGGCCATTGAGCAAACCGCCGATAAGATCTCTGGC1056                           ValGlnIleArgGlnAlaIleGluGlnThrAlaAspLysIleSerGly                               735740745                                                                      ACTGGAACAAACTTCAAGTATGGTAAAATCAACTCAAACAAAGCTGTA1104                           ThrGlyThrAsnPheLysTyrGlyLysIleAsnSerAsnLysAlaVal                               750755760765                                                                   AGATAC1110                                                                     ArgTyr                                                                         (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 370 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        LysGluAlaProLysProPheGlnProIleAsnLysThrLeuAspLys                               151015                                                                         GlyAlaPheGluSerGlyGluValIleValLysPheLysAspGlyVal                               202530                                                                         SerLysLysAlaGlnGlySerAlaLeuAsnLysAlaGluAlaAsnGlu                               354045                                                                         GlnLysAlaSerAlaLysAspProPheGlnValLeuGluValAlaAsp                               505560                                                                         ValAspGlnAlaValLysAlaLeuGluAsnAsnProAsnValGluTyr                               65707580                                                                       AlaGluProAsnTyrThrPheGlnAlaThrTrpSerProAsnAspPro                               859095                                                                         TyrTyrSerAlaTyrGlnTyrGlyProGlnAsnThrSerThrProAla                               100105110                                                                      AlaTrpAspValThrArgGlySerSerThrGlnThrValAlaValLeu                               115120125                                                                      AspSerGlyValAspTyrAsnHisProAspLeuAlaArgLysValIle                               130135140                                                                      LysGlyTyrAspPheIleAspArgAspAsnAsnProMetAspLeuAsn                               145150155160                                                                   GlyHisGlyThrHisValAlaGlyThrValAlaAlaAspThrAsnAsn                               165170175                                                                      GlyIleGlyValAlaGlyMetAlaProAspThrLysIleLeuAlaVal                               180185190                                                                      ArgValLeuAspAlaAsnGlySerGlySerLeuAspSerIleAlaSer                               195200205                                                                      GlyIleArgTyrAlaAlaAspGlnGlyAlaLysValLeuAsnLeuSer                               210215220                                                                      LeuGlyCysGluCysAsnSerThrThrLeuLysSerAlaValAspTyr                               225230235240                                                                   AlaTrpAsnLysGlyAlaValValValAlaAlaAlaGlyAsnAspAsn                               245250255                                                                      ValSerArgThrPheGlnProAlaSerTyrProAsnAlaIleAlaVal                               260265270                                                                      GlyAlaIleAspSerAsnAspArgLysAlaSerPheSerAsnTyrGly                               275280285                                                                      ThrTrpValAspValThrAlaProGlyValAsnIleAlaSerThrVal                               290295300                                                                      ProAsnAsnGlyTyrSerTyrMetSerGlyThrSerMetAlaSerPro                               305310315320                                                                   HisValAlaGlyLeuAlaAlaLeuLeuAlaSerGlnGlyLysAsnAsn                               325330335                                                                      ValGlnIleArgGlnAlaIleGluGlnThrAlaAspLysIleSerGly                               340345350                                                                      ThrGlyThrAsnPheLysTyrGlyLysIleAsnSerAsnLysAlaVal                               355360365                                                                      ArgTyr                                                                         370                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 840 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..840                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                        TGGTCACCGAATGACCCTTACTATTCTGCTTACCAGTATGGACCACAA48                             TrpSerProAsnAspProTyrTyrSerAlaTyrGlnTyrGlyProGln                               375380385                                                                      AACACCTCAACCCCTGCTGCCTGGGATGTAACCCGTGGAAGCAGCACT96                             AsnThrSerThrProAlaAlaTrpAspValThrArgGlySerSerThr                               390395400                                                                      CAAACGGTGGCGGTCCTTGATTCCGGAGTGGATTATAACCACCCTGAT144                            GlnThrValAlaValLeuAspSerGlyValAspTyrAsnHisProAsp                               405410415                                                                      CTTGCAAGAAAAGTAATAAAAGGGTACGACTTTATCGACAGGGACAAT192                            LeuAlaArgLysValIleLysGlyTyrAspPheIleAspArgAspAsn                               420425430                                                                      AACCCAATGGATCTTAACGGACATGGTACCCATGTTGCCGGTACTGTT240                            AsnProMetAspLeuAsnGlyHisGlyThrHisValAlaGlyThrVal                               435440445450                                                                   GCTGCTGATACGAACAATGGAATTGGCGTAGCCGGTATGGCACCAGAT288                            AlaAlaAspThrAsnAsnGlyIleGlyValAlaGlyMetAlaProAsp                               455460465                                                                      ACGAAGATCCTTGCCGTACGGGTCCTTGATGCCAATGGAAGTGGCTCA336                            ThrLysIleLeuAlaValArgValLeuAspAlaAsnGlySerGlySer                               470475480                                                                      CTTGACAGCATTGCCTCAGGTATCCGCTATGCTGCTGATCAAGGGGCA384                            LeuAspSerIleAlaSerGlyIleArgTyrAlaAlaAspGlnGlyAla                               485490495                                                                      AAGGTACTCAACCTCTCCCTTGGTTGCGAATGCAACTCCACAACTCTT432                            LysValLeuAsnLeuSerLeuGlyCysGluCysAsnSerThrThrLeu                               500505510                                                                      AAGAGTGCCGTCGACTATGCATGGAACAAAGGAGCTGTAGTCGTTGCT480                            LysSerAlaValAspTyrAlaTrpAsnLysGlyAlaValValValAla                               515520525530                                                                   GCTGCAGGGAATGACAATGTATCCCGTACATTCCAACCAGCTTCTTAC528                            AlaAlaGlyAsnAspAsnValSerArgThrPheGlnProAlaSerTyr                               535540545                                                                      CCTAATGCCATTGCAGTAGGTGCCATTGACTCCAATGATCGAAAAGCA576                            ProAsnAlaIleAlaValGlyAlaIleAspSerAsnAspArgLysAla                               550555560                                                                      TCATTCTCCAATTACGGAACGTGGGTGGATGTCACTGCTCCAGGTGTG624                            SerPheSerAsnTyrGlyThrTrpValAspValThrAlaProGlyVal                               565570575                                                                      AACATAGCATCAACCGTTCCGAATAATGGCTACTCCTACATGTCTGGT672                            AsnIleAlaSerThrValProAsnAsnGlyTyrSerTyrMetSerGly                               580585590                                                                      ACGTCCATGGCATCCCCTCACGTGGCCGGTTTGGCTGCTTTGTTGGCA720                            ThrSerMetAlaSerProHisValAlaGlyLeuAlaAlaLeuLeuAla                               595600605610                                                                   AGTCAAGGTAAGAATAACGTACAAATCCGCCAGGCCATTGAGCAAACC768                            SerGlnGlyLysAsnAsnValGlnIleArgGlnAlaIleGluGlnThr                               615620625                                                                      GCCGATAAGATCTCTGGCACTGGAACAAACTTCAAGTATGGTAAAATC816                            AlaAspLysIleSerGlyThrGlyThrAsnPheLysTyrGlyLysIle                               630635640                                                                      AACTCAAACAAAGCTGTAAGATAC840                                                    AsnSerAsnLysAlaValArgTyr                                                       645650                                                                         (2) INFORMATION FOR SEQ ID NO:8:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 280 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                        TrpSerProAsnAspProTyrTyrSerAlaTyrGlnTyrGlyProGln                               151015                                                                         AsnThrSerThrProAlaAlaTrpAspValThrArgGlySerSerThr                               202530                                                                         GlnThrValAlaValLeuAspSerGlyValAspTyrAsnHisProAsp                               354045                                                                         LeuAlaArgLysValIleLysGlyTyrAspPheIleAspArgAspAsn                               505560                                                                         AsnProMetAspLeuAsnGlyHisGlyThrHisValAlaGlyThrVal                               65707580                                                                       AlaAlaAspThrAsnAsnGlyIleGlyValAlaGlyMetAlaProAsp                               859095                                                                         ThrLysIleLeuAlaValArgValLeuAspAlaAsnGlySerGlySer                               100105110                                                                      LeuAspSerIleAlaSerGlyIleArgTyrAlaAlaAspGlnGlyAla                               115120125                                                                      LysValLeuAsnLeuSerLeuGlyCysGluCysAsnSerThrThrLeu                               130135140                                                                      LysSerAlaValAspTyrAlaTrpAsnLysGlyAlaValValValAla                               145150155160                                                                   AlaAlaGlyAsnAspAsnValSerArgThrPheGlnProAlaSerTyr                               165170175                                                                      ProAsnAlaIleAlaValGlyAlaIleAspSerAsnAspArgLysAla                               180185190                                                                      SerPheSerAsnTyrGlyThrTrpValAspValThrAlaProGlyVal                               195200205                                                                      AsnIleAlaSerThrValProAsnAsnGlyTyrSerTyrMetSerGly                               210215220                                                                      ThrSerMetAlaSerProHisValAlaGlyLeuAlaAlaLeuLeuAla                               225230235240                                                                   SerGlnGlyLysAsnAsnValGlnIleArgGlnAlaIleGluGlnThr                               245250255                                                                      AlaAspLysIleSerGlyThrGlyThrAsnPheLysTyrGlyLysIle                               260265270                                                                      AsnSerAsnLysAlaValArgTyr                                                       275280                                                                         (2) INFORMATION FOR SEQ ID NO:9:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 679 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                        AAGCTTAATCATCCCGATGTATCGCTTCAGCGCTTACCGTAGACGATTTTCTTTATAGTC60                 TCGATGGATAAAAAGTACCTATCTGAAATGGAAGCAATCGACTCTCCTCCAGTAAAGGCT120                TCAAGGATCGACGTGTTTCTCCGTTTAAGCGCTTCCCTTCCTCCTGACTTTGAGCCCCAG180                GCATTATACTCTTCTTTTCGCTTTGGGATATAAATGGTTTGGCCTTGAACATAGTTTTGT240                AATTCTCTCATTAAGTAATCAGGAATTTGATTTACTGCTTTCAATCGTGTCAGCTCCTTA300                TCATTATTGGATCAATAAGGGACAAAGCCGACATATGAATGGCGATTCATCTAAAACTAC360                CACCCCATGCAAAGGATCGCCGAATCATACAGGCTTTGCATGAGATGCTGCAGATTTCGG420                AAAACGGATTTCCATATGATCACCTCCTAGTATCAGTATACTGATACTAGCAGAAAGATT480                TCCATAAGAATTTCTTATAGTTACCATAATATTATTATATAAACCTACTATATTTGATTT540                TCAATTTGAGAAAATAAGTGACCATTCACCTATCCTTAAAGTTGCTCAACCCCATACAAT600                CATGAAACTTTTCATGCCAAACGTTCATTATGCGAAATCTATCAAAAACTGAGAGTGAAT660                TCATTTTTTGATAGAAAAT679                                                         (2) INFORMATION FOR SEQ ID NO:10:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 81 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..81                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                       ATGAAGTTCAAAAAAATAGCCGCTCTATCCTTAGCAACTTCCCTTGCT48                             MetLysPheLysLysIleAlaAlaLeuSerLeuAlaThrSerLeuAla                               285290295                                                                      TTATTCCCTGCCTTCGGAGGTAGTTCACTGGCC81                                            LeuPheProAlaPheGlyGlySerSerLeuAla                                              300305                                                                         (2) INFORMATION FOR SEQ ID NO:11:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 27 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                       MetLysPheLysLysIleAlaAlaLeuSerLeuAlaThrSerLeuAla                               151015                                                                         LeuPheProAlaPheGlyGlySerSerLeuAla                                              2025                                                                           (2) INFORMATION FOR SEQ ID NO:12:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 873 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..873                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                       ATGAGGAGCTCCCTTGTGCTGTTCTTTGTCTCTGCGTGGACGGCCTTG48                             MetArgSerSerLeuValLeuPhePheValSerAlaTrpThrAlaLeu                               303540                                                                         GCCAGTCCTATTCGTCGAGAGGTCTCGCAGGATCTGTTTAACCAGTTC96                             AlaSerProIleArgArgGluValSerGlnAspLeuPheAsnGlnPhe                               455055                                                                         AATCTCTTTGCACAGTATTCTGCAGCCGCATACTGCGGAAAAAACAAT144                            AsnLeuPheAlaGlnTyrSerAlaAlaAlaTyrCysGlyLysAsnAsn                               60657075                                                                       GATGCCCCAGCTGGTACAAACATTACGTGCACGGGAAATGCCTGCCCC192                            AspAlaProAlaGlyThrAsnIleThrCysThrGlyAsnAlaCysPro                               808590                                                                         GAGGTAGAGAAGGCGGATGCAACGTTTCTCTACTCGTTTGAAGACTCT240                            GluValGluLysAlaAspAlaThrPheLeuTyrSerPheGluAspSer                               95100105                                                                       GGAGTGGGCGATGTCACCGGCTTCCTTGCTCTCGACAACACGAACAAA288                            GlyValGlyAspValThrGlyPheLeuAlaLeuAspAsnThrAsnLys                               110115120                                                                      TTGATCGTCCTCTCTTTCCGTGGCTCTCGTTCCATAGAGAACTGGATC336                            LeuIleValLeuSerPheArgGlySerArgSerIleGluAsnTrpIle                               125130135                                                                      GGGAATCTTAACTTCGACTTGAAAGAAATAAATGACATTTGCTCCGGC384                            GlyAsnLeuAsnPheAspLeuLysGluIleAsnAspIleCysSerGly                               140145150155                                                                   TGCAGGGGACATGACGGCTTCACTTCGTCCTGGAGGTCTGTAGCCGAT432                            CysArgGlyHisAspGlyPheThrSerSerTrpArgSerValAlaAsp                               160165170                                                                      ACGTTAAGGCAGAAGGTGGAGGATGCTGTGAGGGAGCATCCCGACTAT480                            ThrLeuArgGlnLysValGluAspAlaValArgGluHisProAspTyr                               175180185                                                                      CGCGTGGTGTTTACCGGACATAGCTTGGGTGGTGCATTGGCAACTGTT528                            ArgValValPheThrGlyHisSerLeuGlyGlyAlaLeuAlaThrVal                               190195200                                                                      GCCGGAGCAGACCTGCGTGGAAATGGGTATGATATCGACGTGTTTTCA576                            AlaGlyAlaAspLeuArgGlyAsnGlyTyrAspIleAspValPheSer                               205210215                                                                      TATGGCGCCCCCCGAGTCGGAAACAGGGCTTTTGCAGAATTCCTGACC624                            TyrGlyAlaProArgValGlyAsnArgAlaPheAlaGluPheLeuThr                               220225230235                                                                   GTACAGACCGGCGGAACACTCTACCGCATTACCCACACCAATGATATT672                            ValGlnThrGlyGlyThrLeuTyrArgIleThrHisThrAsnAspIle                               240245250                                                                      GTCCCTAGACTCCCGCCGCGCGAATTCGGTTACAGCCATTCTAGCCCA720                            ValProArgLeuProProArgGluPheGlyTyrSerHisSerSerPro                               255260265                                                                      GAGTACTGGATCAAATCTGGAACCCTTGTCCCCGTCACCCGAAACGAT768                            GluTyrTrpIleLysSerGlyThrLeuValProValThrArgAsnAsp                               270275280                                                                      ATCGTGAAGATAGAAGGCATCGATGCCACCGGCGGCAATAACCAGCCT816                            IleValLysIleGluGlyIleAspAlaThrGlyGlyAsnAsnGlnPro                               285290295                                                                      AACATTCCGGATATCCCTGCGCACCTATGGTACTTCGGGTTAATTGGG864                            AsnIleProAspIleProAlaHisLeuTrpTyrPheGlyLeuIleGly                               300305310315                                                                   ACATGTCTT873                                                                   ThrCysLeu                                                                      (2) INFORMATION FOR SEQ ID NO:13:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 291 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                       MetArgSerSerLeuValLeuPhePheValSerAlaTrpThrAlaLeu                               151015                                                                         AlaSerProIleArgArgGluValSerGlnAspLeuPheAsnGlnPhe                               202530                                                                         AsnLeuPheAlaGlnTyrSerAlaAlaAlaTyrCysGlyLysAsnAsn                               354045                                                                         AspAlaProAlaGlyThrAsnIleThrCysThrGlyAsnAlaCysPro                               505560                                                                         GluValGluLysAlaAspAlaThrPheLeuTyrSerPheGluAspSer                               65707580                                                                       GlyValGlyAspValThrGlyPheLeuAlaLeuAspAsnThrAsnLys                               859095                                                                         LeuIleValLeuSerPheArgGlySerArgSerIleGluAsnTrpIle                               100105110                                                                      GlyAsnLeuAsnPheAspLeuLysGluIleAsnAspIleCysSerGly                               115120125                                                                      CysArgGlyHisAspGlyPheThrSerSerTrpArgSerValAlaAsp                               130135140                                                                      ThrLeuArgGlnLysValGluAspAlaValArgGluHisProAspTyr                               145150155160                                                                   ArgValValPheThrGlyHisSerLeuGlyGlyAlaLeuAlaThrVal                               165170175                                                                      AlaGlyAlaAspLeuArgGlyAsnGlyTyrAspIleAspValPheSer                               180185190                                                                      TyrGlyAlaProArgValGlyAsnArgAlaPheAlaGluPheLeuThr                               195200205                                                                      ValGlnThrGlyGlyThrLeuTyrArgIleThrHisThrAsnAspIle                               210215220                                                                      ValProArgLeuProProArgGluPheGlyTyrSerHisSerSerPro                               225230235240                                                                   GluTyrTrpIleLysSerGlyThrLeuValProValThrArgAsnAsp                               245250255                                                                      IleValLysIleGluGlyIleAspAlaThrGlyGlyAsnAsnGlnPro                               260265270                                                                      AsnIleProAspIleProAlaHisLeuTrpTyrPheGlyLeuIleGly                               275280285                                                                      ThrCysLeu                                                                      290                                                                            (2) INFORMATION FOR SEQ ID NO:14:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 9 amino acids                                                      (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                       AlaTyrGlnTyrGlyProGlnAsnThr                                                    15                                                                             (2) INFORMATION FOR SEQ ID NO:15:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 12 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                       TyrAspPheIleAspTyrAspAsnAsnProMetAsp                                           1510                                                                           (2) INFORMATION FOR SEQ ID NO:16:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                       GCTACATAGGCCCAAAAC18                                                           (2) INFORMATION FOR SEQ ID NO:17:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 25 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                       TCCATGGTTTTTCTATCATAATCTA25                                                    (2) INFORMATION FOR SEQ ID NO:18:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 628 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                       GAATTCGGCTTAAGGCATGCGTCCTTCTTTGTGCTTGGAAGCAGAGCCCAATATTATCCC60                 GAAACGATAAAACGGATGCTGAAGGAAGGAAACGAAGTCGGCAACCATTCCTGGGACCAT120                CCGTTATTGACAAGGCTGTCAAACGAAAAAGCGTATCAGGAGATTAACGACACGCAAGAA180                ATGATCGAAAAAATCAGCGGACACCTGCCTGTACACTTGCGTCCTCCATACGGCGGGATC240                AATGATTCCGTCCGCTCGCTTTCCAATCTGAAGGTTTCATTGTGGGATGTTGATCCGGAA300                GATTGGAAGTACAAAAATAAGCAAAAGATTGTCAATCATGTCATGAGCCATGCGGGAGAC360                GGAAAAATCGTCTTAATGCACGATATTTATGCAACGTTCGCAGATGCTGCTGAAGAGATT420                ATTAAAAAGCTGAAAGCAAAAGGCTATCAATTGGTAACTGTATCTCAGCTTGAAGAAGTG480                AAGAAGCAGAGAGGCTATTGAATAAATGAGTAGAAAGCGCCATATCGGCGCTTTTCTTTT540                GGAAGAAAATATAGGGAAAATGGTACTTGTTAAAAATTCGGAATATTTATACAATATCAT600                ATGTTACACATTGAAAGAAGCCGAATTC628                                                (2) INFORMATION FOR SEQ ID NO:19:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 16 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                       AAGGCATGCGTCCTTC16                                                             (2) INFORMATION FOR SEQ ID NO:20:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                       CTTTCAATGTGTAACATA18                                                           (2) INFORMATION FOR SEQ ID NO:21:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 1514 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                       GAATTCGGCTTGAGACCCGGGAGCTTTCAGTGAAGTACGTGATTATACGGAGATGAAAAT60                 TCGTACACTGTTAACGAGAAGGAAACGCCGACGAAAGCGTAGCATCGGATGGCAAAGATG120                GAGTAACGAATATCTCTACGGTGTACTGGGGCTTTACTGAGACTAGAAAGTCCTTCCCTT180                GAAAAGTGCAGAGAGTTTTCGATAAAAGTGTCAGCCATTTGATAAGTCTCATTCTCATAA240                CCTATTGATGAAGTTTATAGGGAAGCTGCTTGAGAGGGAAAACCTCACGAACAGTTCTTA300                TGGGGAGAGACTGGAAACAGGTCACAATTGATACCTCGCTAATCTTTTAACCGACAAAGT360                TTTTTTAAACCGTGGAAGTCATAATAACCTGGATATTGTGAATTTATAAAAGTTAACAAA420                TGGTTTATATTAAGACAGTCATAAACCAAAGATTTTTCTTCTAAAGCTACGATAGCAAAA480                ATTTCACTAGAAATTAGTTATACAAGCATTTTGTAAGAATTATTAAAAAGATAAATCCTG540                CTATTACGAGATTAGTAGGATGATATTGTGAAAAATTTTTTATCTATTCGATTTAAAATA600                TTTATGAATTTTACATAAACCTCATAAGAAAAAATACTATCTATACTATTTTAAGAAATT660                TATTAGAATAAGCGGATTCAAAATAGCCCTGGCCATAAAAGTACCTCAGCAGTAGAAGTT720                TTGACCAAAATTAAAAAAATACCCAATCAAGAGAATATTCTTAATTACAATACGTTTTGC780                GAGGAACATATTGATTGAAATTTAATAAATTTAGTCCTAAAATTTAAAGAAATTTAAGTT840                TTTCATATTTTTATGAACTAACAAGAATAAAAATTGTGTTTATTTATTATTCTTGTTAAA900                TATTTGATAAAGAGATATATTTTTGGTCGAAACGTAAGATGAAACCTTAGATAAAAGTGC960                TTTTTTTGTTGCAATTGAAGAATTATTAATGTTAAGCTTAATTAAAGATAATATCTTTGA1020               ATTGTAACGCCCCTCAAAAGTAAGAACTACAAAAAAAGAATACGTTATATAGAAATATGT1080               TTGAACCTTCTTCAGATTACAAATATATTCGGACGGACTCTACCTCAAATGCTTATCTAA1140               CTATAGAATGACATACAAGCACAACCTTGAAAATTTGAAAATATAACTACCAATGAACTT1200               GTTCATGTGAATTATCGCTGTATTTAATTTTCTCAATTCAATATATAATATGCCAATACA1260               TTGTTACAAGTAGAAATTAAGACACCCTTGATAGCCTTACTATACCTAACATGATGTAGT1320               ATTAAATGAATATGTAAATATATTTATGATAAGAAGCGACTTATTTATAATCATTACATA1380               TTTTTCTATTGGAATGATTAAGATTCCAATAGAATAGTGTATAAATTATTTATCTTGAAA1440               GGAGGGATGCCTAAAAACGAAGAACATTAAAAACATATATTTGCACCGTCTAATGGATTT1500               ATGAAGCCGAATTC1514                                                             (2) INFORMATION FOR SEQ ID NO:22:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 30 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                       GAGACCCGGGAGCTTTCAGTGAAGTACGTG30                                               (2) INFORMATION FOR SEQ ID NO:23:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                       CATAAATCCATTAGACGGTGC21                                                        (2) INFORMATION FOR SEQ ID NO:24:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 162 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                       ATCGATTGTTTGAGAAAAGAAGAAGACCATAAAAATACCTTGTCTGTCATCAGACAGGGT60                 ATTTTTTATGCTGTCCAGACTGTCCGCTGTGTAAAAATAAGGAATAAAGGGGGGTTGTTA120                TTATTTTACTGATATGTAAAATATAATTTGTATAAGAAAATG162                                  (2) INFORMATION FOR SEQ ID NO:25:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 29 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                       GCATGCAATCGATTGTTTGAGAAAAGAAG29                                                (2) INFORMATION FOR SEQ ID NO:26:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                       CATTTTCTTATACAAATTATATTTTACATATCAG34                                           (2) INFORMATION FOR SEQ ID NO:27:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 2017 base pairs                                                    (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                       TGCGAAATCTATCAAAAACTGAGAGTGAATTCATTTTTTGATAGAAAATTAAAACTATTC60                 AATATTTTGTCACAACCTGCTAGAATCCTAGGTAATAAGGGTCCCCTACATATCTATCAT120                TCATCACAATGACCTTTGTTCATCTTGAATTCTGAAGGGAGGATCGACCTGCTAATTTGT180                CGTAAAAAAATAGAAAATGGAGGAATGCTTTTATGAAGTTCAAAAAAATAGCCGCTCTAT240                CCTTAGCAACTTCCCTTGCTTTATTCCCTGCCTTCGGAGGTAGTTCACTGGCCAAGGAAG300                CACCGAAACCGTTCCAACCTATCAACAAAACTTTGGATAAAGGCGCTTTCGAATCCGGTG360                AAGTCATCGTCAATTCAAAGATGGTGTATCCAAAAAGGCACAAGGTTCTGCTCTGAACAA420                AGCGGAGGCAAATGAACAGAAAGCATCAGCAAAAGATCCATTTCAGGTATTGGAAGTAGC480                GGACGTCGATCAAGCTGTTAAAGCACTGGAAAACAATCCGAATGTAGAATATGCTGAACC540                AAACTATACCTTCCAAGCGACTTGGTCACCGAATGACCCTTACTATTCTGCTTACCAGTA600                TGGACCACAAAACACCTCAACCCCTGCTGCCTGGGATGTAACCCGTGGAAGCAGCACTCA660                AACGGTGGCGGTCCTTGATTCCGGAGTGGATTATAACCACCCTGATCTTGCAAGAAAAGT720                AATAAAAGGGTACGACTTTATCGACAGGGACAATAACCCAATGGATCTTAACGGACATGG780                TACCCATGTTGCCGGTACTGTTGCTGCTGATACGAACAATGGAATTGGCGTAGCCGGTAT840                GGCACCAGATACGAAGATCCTTGCCGTACGGGTCCTTGATGCCAATGGAAGTGGCTCACT900                TGACAGCATTGCCTCAGGTATCCGCTATGCTGCTGATCAAGGGGCAAAGGTACTCAACCT960                CTCCCTTGGTTGCGAATGCAACTCCACAACTCTTAAGAGTGCCGTCGACTATGCATGGAA1020               CAAAGGAGCTGTAGTCGTTGCTGCTGCAGGGAATGACAATGTATCCCGTACATTCCAACC1080               AGCTTCTTACCCTAATGCCATTGCAGTAGGTGCCATTGACTCCAATGATCGAAAAGCATC1140               ATTCTCCAATTACGGAACGTGGGTGGATGTCACTGCTCCAGGTGTGAACGAGGTTACTAG1200               CTTTTCGTAGTAAGAGGTTAATGCCTTGCACCCACCTACAGTGACGAGGTCCACACTTGA1260               TAGCATCAACCGTTCCGAATAATGGCTACTCCTACATGTCTGGTACGTCCATGGCATCCC1320               CTCACGTGGCCGGTTTGGCTGCTTTGTTGGCAAGTCAAGGTAAGAATAACGTACAAATCC1380               GCCAGGCCATTGAGCAAACCGCCGATAAGATCTCTGGCACTGGAACAAACTTCAAGTATG1440               GTAAAATCAACTCAAACAAAGCTGTAAGATACTAATAGATAAAACAAGAGCACACCGTGA1500               ATGGTGGGCTCTTTCATTATGTTCACTACTGTTTTACGATCTGGCCGTTTTGGTTCAGGT1560               AAACACTCTGGATGATGGTTCTATTAAACGGTTTCCCTTTATAATCAGACTTAATATCCG1620               TTGTCAGGTTGTAGGTTCCTTCTCCTCCATTGAACACTGTACCACTCCCCTTGACAGACA1680               ATTATAGGCAACAGTCCAACATCCAAGGAAGAGGAGGTAACTTGTGACATGGTGAGGGGA1740               ACTGTCTGTGGGACAAAGGTTTCCCCTTAGGGTAGAACTCAAACTTGTGTGCTCGGTGAA1800               CCCACTGACGATACTTGACCCTGTTTCCAAAGGGGAATCCCATCTTGAGTTTGTAACACA1860               CGAGCCACTTGGGTGACTGCTATGAACTAACTTGCGACTGAGGGAAAGAGTCACCAGCGT1920               GGTGGTTCAGTAGTAAGTTACCTGAACACTTTGGTTCATTCAGTTTTGCCCCCAGGTTCT1980               GGGAAAAAGGATGAACTTCCACTTCGGCCCTTTTTTT2017                                      __________________________________________________________________________ 

What is claimed is:
 1. A recombinant method for producing an alkaline protease in a microbial host cell comprising the following steps:(a) transforming said host cell with a vector comprising a nucleic acid construct comprising a nucleic acid sequence encoding an alkaline protease, wherein said alkaline protease-encoding sequence comprises a region encoding a mature protease having the amino acid sequence of SEQ ID NO:8 and a promoter operably linked with said nucleic acid construct, said promoter comprising a transcription activating region of the nucleic acid sequence set forth in SEQ ID NO:9; (b) culturing the transformed host cell of step (a); and, (c) recovering said alkaline protease from the transformed host cell of step (b) or from the culture medium of said host cell.
 2. The method according to claim 1 wherein said alkaline protease-encoding sequence has the nucleic acid sequence set forth in SEQ ID NO:7.
 3. A recombinant method for producing an alkaline protease in a microbial host cell comprising the following steps:(a) transforming said host cell with a vector comprising a nucleic acid construct comprising a nucleic acid sequence encoding an alkaline protease, wherein said alkaline protease-encoding sequence comprises a region encoding a protease having the amino acid sequence of SEQ ID NO:6, a signal peptide-encoding sequence, and a promoter operably linked with said nucleic acid construct, said promoter comprising a transcription activating region of the nucleic acid sequence set forth in SEQ ID NO:9; (b) culturing the transformed host cell of step (a); and, (c) recovering said alkaline protease from the transformed host cell of step (b) or from the culture medium of said host cell.
 4. The method according to claim 3 in which the signal peptide-encoding nucleic acid sequence encodes the amino acid sequence set forth in SEQ ID NO:11.
 5. The method according to claim 3 wherein said alkaline protease-encoding sequence has the nucleic acid sequence set forth in SEQ ID NO:5.
 6. A recombinant method for producing an alkaline protease in a microbial host cell comprising the following steps:(a) transforming said host cell with a vector comprising a nucleic acid construct comprising a nucleic acid sequence encoding an alkaline protease, wherein said alkaline protease-encoding sequence comprises a region encoding a protease having the amino acid sequence of SEQ ID NO:4 and a promoter operably linked with said nucleic acid construct, said promoter comprising a transcription activating region of the nucleic acid sequence set forth in SEQ ID NO:9; (b) culturing the transformed host cell of step (a); and, (c) recovering said alkaline protease from the transformed host cell of step (b) or from the culture medium of said host cell.
 7. The method according to claim 1 in which said nucleic acid construct comprises the nucleic acid sequence set forth in SEQ ID NO:1.
 8. The method according to claim 6 wherein said alkaline protease-encoding sequence has the nucleic acid sequence set forth in SEQ ID NO:3. 